While much emphasis in recent years has been focused on the identification and dissection of signaling pathways mediated by protein kinase cascades, it has long been recognized that protein dephosphorylation plays an essential role in regulating the activity of many enzymes involved in cell growth and metabolism (Krebs E. G. and Fischer E. H., Biochim. Biophys. Acta, 1953;20:150; Shenolikar S.,Annu. Rev. Cell Biol., 1994;10:55; Saltiel A. R., FASEB J., 1994;8:1034). Studies in evolutionarily distant organisms have demonstrated a critical role for type 1 protein phosphatase (PP1) action in modulating a wide variety of intracellular processes. In yeast, PP1 has been shown regulate cell cycle progression (Hisamoto N., et al., Mol. Cell. Biol. 1994; 14:3158; Zang S., et al., Mol. Cell. Biol., 1995;15:2037), chromosome segregation (Francisco L., et al., Mol. Cell. Biol., 1994;14:4731), protein synthesis (Wek R. C., et al., Mol. Cell. Biol., 1992;12:5700), and glycogen metabolism (Cannon J. F., et al., Genetics, 1994;136:485). In mammalian cells, PP1 also has multiple physiological roles, such as regulation of glycogen metabolism (Bollen M. and Stalmans W., Crit. Rev. Biochem. Mol. Bio., 1992;27:227), protein synthesis (Cohen P., Ann. Rev. Biochem., 1989;58,453), and muscle contraction (Shenolikar S., Annu. Rev. Cell Biol., 1994;10:55). Many of the metabolic effects of insulin are thought to occur via activation of PP1 (Saltiel A. R., Am. J. Physiol., 1996;33:E375). Indeed, many of the rate-limiting enzymes involved in glucose and lipid metabolism, such as glycogen synthase, hormone sensitive lipase, and pyruvate dehydrogenase are regulated by dephosphorylation. Thus, these dephosphorylations are likely to be critical to many of the metabolic effects of insulin, including stimulation of glycogen and lipid synthesis, and inhibition of lipolysis. Given the array of physiological processes presumed to be mediated by PP1, it becomes apparent that organisms must have evolved a mechanism of regulating PP1 activity to maintain substrate specificity and ensure against accidental activation of competing signaling pathways.
Early biochemical studies on the regulation of protein phosphatases lead to the hypothesis that protein phosphatases acted to constitutively oppose the action of specific protein kinases, since purified phosphatases could act on a wide variety of phosphorylated substrates in vitro. This idea has been challenged recently by the identification of tissue specific proteins that act to target Ser/Thr phosphatases to specific subcellular locations, thereby endowing phosphatases with a high degree of specificity in vivo (Hubbard M. J. and Cohen P., Trends Biochem. Sci., 1993;18:172; Mochly-Rosen D., Science, 1995;268:247). For example, in striated muscle, two targeting subunits M and G, direct the catalytic subunit of PP1, PP1C, to different subcellular locations. The M subunit directs PP1C to myofibrils, acting to facilitate dephosphorylation of myosin (Dent P., et al., Eur. J. Biochem., 1992;210:1037), whereas the G subunit localizes PP1C to both the glycogen particle and the membranes of the sarcoplasmic reticulum, where glycogen metabolizing enzymes and SR proteins serve as substrates for PP1C (Stralfors P., et al., Eur. J. Biochem., 1985;149:295; Hubbard M. J. and Cohen P., Eur. J. Biochem., 1989;186:71 1; Hubbard M. J. and Cohen P., Eur. J. Biochem., 1990;189:243; Macdougall L. K., et al., Eur. J. Biochem., 1991;196:725). In addition, PP1C is also known to interact with proteins in nuclei, an inhibitor protein, NIPP1 (Beullins M., et al., J. Biol. Chem., 1992;267:16538), and the Saccharomyces cerevisiae protein sds22.sup.+ (Stone E. M., et al., Curr. Biol., 1993;3:13). PP1C has also been shown to interact with the product of the tumor suppressor gene, Rb, implicating PP1C in the control of tumorigenesis and cell cycle progression (Durfee T., et al., Genes Dev., 1993;7:555). Thus, the targeting of PP1C to discrete subcellular locations by physically interacting proteins allows for a high degree of substrate specificity and tight control of phosphatase activity.
Recently, a number of proteins that direct PP1C to the glycogen pellet have been characterized and cloned from mammals and yeast. In Saccharomyces cerevisiae, the product of the GAC1 gene is required for glycogen metabolism and physically interacts with PP1C (Stuart J. S., et al., Mol. Cell. Biol., 1994;14:896). In mammals, two tissue specific glycogen localizing subunits of PP1C have been identified. RG1, the glycogen binding subunit of skeletal muscle, encodes a protein product of 160 kD and is expressed in both heart and skeletal muscle (Tang P. M., et al., J. Biol. Chem., 1991;266:15782). Reversible phosphorylation on two closely spaced serine residues contained within the amino terminal portion of RG1 (sites 1 and 2) has been implicated in regulating PP1C activity in response to hormonal stimulation (Dent P., et al., Nature, 1990;348:302). According to this hypothesis, phosphorylation of site 1 following insulin stimulation leads to a higher affinity of PP1C for RG1, leading to activation of glycogen metabolizing enzymes by dephosphorylation, while phosphorylation of site 2 by cAMP activated protein kinase A (PKA) causes a reduced affinity and subsequent release of PP1 C from the glycogen pellet. A glycogen binding subunit expressed exclusively in liver, G.sub.L, was recently cloned and was found to encode a predicted protein product of only 33 kD. G.sub.L was shown to also differ from RG1 with respect to enzymatic activity towards various substrates and in the ability of G.sub.L to serve as a substrate for PKA (Doherty M. J., et al., FEBS Lett., 1995;375:294).
In an effort to identify novel PP1C localizing subunits involved in regulating insulin stimulated metabolic pathways, a 3T3-L1 adipocyte cDNA 2-hybrid library was screened for PP1C interacting proteins. We describe the isolation and characterization of a novel glycogen binding subunit of PP1C, called PTG, that may act as a scaffold for the localization of critical enzymes in glycogen metabolism, including phosphorylase b, glycogen synthase, and phosphorylase kinase. PTG is expressed predominantly in insulin-sensitive tissues and was found to mediate the hormonal control of glycogen accumulation in intact cells.